Our ongoing investigation of frameshift mutagenesis in the T4 rII system reveals the specific enhancement of particular pathways of spontaneous frameshift mutagenesis by different T4 DNA polymerases. Our studies now permit us to classify certain frameshift mechanisms by the genetic outcome (the nature of the DNA sequence change) and thus to examine the role of DNA polymerase defects exhibition by mutator and antimutator T4 DNA polymerases. For example, the mutator polymerase tsL98 produces a large increase in frameshift mutations occurring in runs of A:T base pairs (100 to 200-fold) while producing no increase in the addition of single base pairs not occurring in mononucleotide runs. Taken as a whole, our data strongly support the notion that fremeshifts of these two types occur via different mechanisms. Sequences of spontaneous frameshifts offer support for two novel mechanisms of frameshift mutation. The first mechanism depends upon the correction of imperfect palindromic DNA sequences to more perfect palindromic DNA sequences. The second mechanism generates frameshifts by converting an imperfectly homologous sequence to a sequence that is perfectly homologous, but lies 256 base pairs down stream. This intrageneic conversion event occurs at a substantial frequency in certain DNA polymerase mutant backgrounds examined. Sequences of proflavin-induced frameshift mutation in a wild type polymerase background demonstrate strong sequence specificity. Our initial results identify 14/16 mutants lie within a six base pair sequence. Not all of the mutations are identical (4 distinct genotypes have been observed). This demonstrates a direct sequence effect on occurence of frameshift mutations but may suggest the possibility of additional factors that define the specificity of these frameshift sites. Further studies in different mutant backgrounds and with other intercalating agents should permit a distinction between elements of site specificity dependent on the site of preferred mutagen binding from site specificity dependent upon site-specific DNA metabolic events.